Isolation of a serine Kunitz trypsin inhibitor from leaves of Terminalia arjuna

Abstract

A serine Kunitz protease inhibitor was isolated from the semi-mature leaves of Terminalia arjuna, a host plant for Antheraea mylitta, using ammonium sulphate fractionation, gel permeation chromatography and trypsin–sepharose affinity chromatography. A 29-fold purification of T. arjuna Trypsin Inhibitor (TaTI) with a yield recovery of 3.2% was achieved. The purified protease inhibitor (TaTI) was resolved into a single protein band corresponding to molecular weight of 19.0 kDa on 12% SDS–PAGE under non-reducing conditions, whereas an additional band of 21.5 kDa was observed when the same fraction was resolved on SDS–PAGE under reducing conditions in the presence of 2-mercaptoethanol. TaTI inhibited both trypsin and chymotrypsin, but showed higher affinity for trypsin compared to chymotrypsin. However, it is more effective on bovine trypsin than midgut trypsin of tasar silkworm. TaTI retains its activity over a wide range of temperatures (0–100°C) and pH (2.0–8.0), with pH optimum of 8.0. These observations indicate that TaTI is not only specific to tasar silkworm but also to bovine serine proteases. Hence it can be considered as a generalist protease inhibitor.